Gene engineer strain of Aspergillus niger with high L-malic acid yield, and construction and application thereof

A technology of genetically engineered strains, Aspergillus niger, applied in the direction of genetic engineering, application, plant genetic improvement, etc., can solve problems such as low malic acid yield

Active Publication Date: 2019-01-15
南京昊禾生物科技有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, due to the low malic acid yield of wild-type Aspergillus niger strains, few studies have reported the transforma

Method used

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  • Gene engineer strain of Aspergillus niger with high L-malic acid yield, and construction and application thereof
  • Gene engineer strain of Aspergillus niger with high L-malic acid yield, and construction and application thereof
  • Gene engineer strain of Aspergillus niger with high L-malic acid yield, and construction and application thereof

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Experimental program
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Embodiment 1

[0042] This embodiment includes the following steps:

[0043] (1) Construction of plasmid containing loxP-hph-loxP element

[0044] The loxP-hph-loxP element sequence was synthesized by Beijing Huada Gene, and then ligated and cloned into the vector pFGL59Ble that had undergone the same double digestion to construct plasmid pLH314 (loxP-hph-loxP, ble r ). pLH314 was digested with Xho I and ligated to obtain plasmid pLH331(loxP-hph-loxP). pLH314 is used to construct the starting vector of the knockout plasmid; use the Aspergillus niger ATCC1015 genome as a template, and p635 / p636 and p637 / p638 as primers to amplify the DNA sequences at both ends of the pyrG gene, and then clone them into the vector pLH314 to construct the pyrG knockout plasmid pLH323 .

[0045] (2) Construction of plasmid containing Tet-on-cre element

[0046] The hph resistance gene was removed by cutting the vector pFGL59 with Sal I, and the vector fragment was recovered and ligated with DNAT4 ligase to o...

Embodiment 2

[0054] This embodiment includes the following steps:

[0055] (1) Construction of pyc expression plasmid

[0056] To amplify the pyc gene sequence, design primers p994 / p995 (Table 1), amplify the pyc gene sequence fragment by PCR, send it to Huada Gene Company for sequencing to confirm that there is no mutation, and then recover after Spe I and Hind III double enzyme digestion Ligate with the plasmid fragment pLH331 that was also digested with the same enzyme, transform the ligated product into Escherichia coli JM109 competent cells, spread evenly on LB culture dishes containing 100 μg / mL kanamycin, culture overnight at 37°C, and pick Take a single clone, and verify it by colony PCR and double enzyme digestion ( figure 2 ) to obtain the pyc expression plasmid pLH395.

[0057] The gene pyc sequence includes the promoter and terminator of the gene itself, which is SEQ NO.1 in the sequence listing, and the length is 5247bp.

[0058] (2) Construction of mdh expression plasmid ...

Embodiment 3

[0074] This embodiment includes the following steps:

[0075] Agrobacterium-mediated transformation of Aspergillus niger and clone screening: the overexpression or heterologous expression described in the present invention refers to the integration of related genes into the genome of Aspergillus niger for expression. The transformation method of expressing gene and knocking out gene described in the present invention is Agrobacterium-mediated method (Chen et al. 2014, reference 1). The Agrobacterium is AGL-1 strain. Before the expression gene and the knockout gene of the present invention are transformed into Aspergillus niger mediated by Agrobacterium, the expression plasmid and the knockout plasmid need to be electrotransformed into Agrobacterium first. The electroporation conditions are: Capacitnce: 25uF, Voltage: 2.5kV, Resistance: 200Ω, Pulse: 5msec.

[0076] (1) Obtaining of pyc gene overexpression strain

[0077] The plasmid pLH395 was electrotransferred into Agrobac...

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Abstract

The invention provides a gene engineer strain of Aspergillus niger with high L-malic acid yield and a construction method thereof. The method utilizes gene knockout technique to disrupt the Aspergillus niger oxaloacetic acid hydrolase gene (oahA) so as to obtain the malic acid-producing strain S2. After 7 days of shake flask fermentation, 100 g/L glucose is converted into 120.4 +/- 2. 8 g/L of L-malic acid, the conversion of malic acid and malic acid to glucose is 1.62 mol/mol, which is 81% of the highest conversion in theory. A good strain for the industrialized production of malic acid is provided.

Description

technical field [0001] The invention belongs to the field of bioengineering, and more specifically relates to the construction and application of a high-yield L-malic acid Aspergillus niger genetic engineering strain. Background technique [0002] L-malic acid, also known as 2-hydroxysuccinic acid, is mainly used in food, medicine and other industries. In the food industry, because L-malic acid has a soft taste, high acidity, and does not damage the oral cavity and teeth, and does not accumulate fat, it has become a low-calorie, safe food sour agent recognized by the international food industry. It is currently the largest amount used in the world's food industry. And one of the organic acids with better development prospects. In the pharmaceutical industry, L-malic acid is used to treat various diseases such as liver disease, anemia, and uremia. And because L-malic acid is beneficial to the absorption of amino acids in metabolism, it is often formulated into compound amin...

Claims

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Application Information

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IPC IPC(8): C12N1/15C12N15/80C12N15/90C12N15/52C12N15/53C12N15/55C12N15/31C12P7/46C12R1/685
CPCC07K14/38C12N9/0006C12N9/14C12N9/93C12N15/80C12N15/902C12N2800/30C12P7/46C12Y101/01037C12Y307/01001C12Y604/01001
Inventor 刘浩徐永学
Owner 南京昊禾生物科技有限公司
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